Serologic test for therapy control of HPV16 positive carcinoma

ABSTRACT

A method for therapy control of HPV16 positive carcinoma, an antibody for use in the corresponding diagnostic method as well as a test for performing the method. In particular, a serologic method for monitoring the development of the amount of antibodies in samples, which were taken from a patient before and after the treatment of a HPV16 positive carcinoma over a predetermined period of time. In addition, an immunologic test in the form of a kit, with which the method can be performed.

BACKGROUND Technical Field

The present disclosure relates to a method for therapy control of HPV16positive carcinoma, an antibody for use in the corresponding diagnosticmethod as well as a test for performing the method.

Description of the Related Art

Meanwhile, more than 100 types of human papilloma viruses (HPV) areknown, which can infect the epithelial cells of the skin or of variousmucosae. HPV infections are widely spread and different HPV types areattributed to different clinical pictures. HPV of type 1 and HPV of type2 cause warts on the hands and feet, while HPV of type 6 and HPV of type11 genital warts. In many cases, such an infection has no clinicalsymptoms, but it can also result in a tumor-like growth of the affectedepithelial cells. Even though such tumors are mostly benign and, asmentioned above, only lead to the formation of warts, it has meanwhilebeen established that some HPVs can also cause malignant changes andtherefore be responsible for the development of cancer, for example inthe genital region but also in the mouth or throat.

Therapy method of choice for these malignant tumors is surgery,radiotherapy, chemotherapy, immunotherapy or a combination of thesemethods. In the context of therapy control after treatment of HPVpositive carcinoma, it is desirable to detect tumor cells remaining inthe body but also a relapse or metastases early, so that treatment canbe resumed, for example by chemotherapy or immunotherapy, before visiblesecondary tumors are formed.

Various publications deal with the determination of HPV specificantibodies in the serum of patients and the diagnostic and prognosticvalue of the obtained data with respect to the occurrence orreoccurrence of HPV positive carcinoma.

Particularly useful for deducing diagnostic or prognostic values areso-called tumor antigens, i.e., particular antigen structures, that arepart of a tumor cell and specific for it, and which are recognized bythe immune system and can cause an immune response. The so-called tumorantigens HPV E6 and HPV E7 are only suitable to a limited extent,however, because these protein antigens occur in all HPV types, arelargely homologous and therefore do not allow a type specificassessment, not even when, for example, HPV16 specific E6 or E7 proteinsare used for serologic detection. This type specific detection of theserologic response is necessary, however, to determine the reaction ofthe immune system to the HPV type, which caused the tumor, and not, forexample, the immune response to a benign wart on the foot, which wouldbe considered a false-positive result and could have fatal consequencesfor the patient.

HPV are dsDNA viruses. The non-encapsulated virions consist oficosahedral capsids. L1 (late protein 1) determines amongst other thingsthe capsid formation of the HPV and is primarily responsible for theimmunogenicity of HPV types.

Af Geijerstam et al. describe in Journal of Infectious Diseases, 177,1998, 1710-1714 a study, in which serum levels of HPV16 capsid specificantibodies in primiparous women were determined over a period of timeuntil the second pregnancy. It can be inferred from this study, that theamount of HPV16 capsid specific antibodies in the serum remains stableover several years and the amount of antibodies correlates with thenumber of sexual partners, but not with a medical condition.

A further study deals with the question, whether HPV16 infectionsrepresent a risk factor for the later occurrence of cervix carcinoma(Shah et al., Cancer Epidemiology, Biomarkers & Prevention, 6, 1997,233-237). The presence of a larger amount of HPV16 capsid antibodies inthe serum is associated with a higher risk for the occurrence of cervixcarcinoma. It was also found in the tests that the HPV16 capsidantibodies did not recede noticeably over a period of 7 to 13 years.

Koslabova et al. stated in International Journal of Cancer, 133, 2013,1832-1839, that a long lasting seropositivity against HPV16 virus likeparticles (VLPs), i.e., capsids, is observed after the therapy of tumorpatients. This means that a decrease of the amount of L1 specificantibodies after therapy does not appear suitable to control the successof the therapy. It is mentioned in the summary, that the titer of theantibodies, which are specific for the HPV16 capsid antigens, does notchange during the observation period after treatment. L1 therefore doesnot represent a marker, which might be suitable for monitoring thedevelopment or even to detect a relapse.

A further problem as already mentioned above is the lack of typespecificity of the conventional antibody tests. L1 or also the maincapsid protein of HPV can be in monomeric or multimeric form. Fivesingle L1 proteins associate to build so-called capsomers (orpentamers). 72 capsomers, as sub-units of the capsids, associate to formthe capsid of the viruses, in which the genetic material is packedduring naturally occurring infection. Differences in the nucleic acidsequence of 10% within the L1 gene are defined as a requirement in orderto describe a new HPV type. This means, that even different HPV typesmay be identical in almost 90% of the L1 gene and protein. Besides themain capsid protein (L1), there is also the minor capsid protein L2. TheL2 protein is also a highly conserved, i.e., in large parts identicalprotein. The L2 protein is therefore also not specific for particularHPV types.

Nevertheless, there can be parts in both proteins, which are specificfor particular HPV types (see, e.g., Christensen). That means, thattype-specific and non-type-specific (group-specific e.g., for high risktypes or also genus-specific) epitopes (binding sites for antibodies)may occur next to each other.

As the antibodies of the patient sample are of polyclonal origin, i.e.,directed against many different antigens or different parts of anantigen, type-specific and non-type-specific L1 antibodies cannot bedistinguished when using monomeric L1 proteins in a “traditional”arrangement of an ELISA test with peroxidase or fluorescent labeledanti-IgG specific conjugates. Merely the presence of anti-L1 antibodiesof the more than 100 different HPV is detected, even if the L1 proteinis derived from HPV type 16. Because of the very high homology of theprotein within the group of HPV, the seeming type specificity of theused L1 antigen is very misleading.

As a result, HPV serology is not suitable for therapy control of HPV16positive carcinoma because it is accepted in the state of the art, thatthe amount of antibodies remains stable over years and does notnecessarily decrease after therapy, so that this parameter is notsuitable for a monitoring the condition. Furthermore, the different HPVtypes cannot be distinguished using conventional antibody tests.

BRIEF SUMMARY

Surprisingly, it was found out now, that for particular HPV16 L1 capsidspecific antibodies, which bind to at least one conformational epitopeof the HPV16 L1 capsid, which is not present in monomeric and/ordenatured HPV16 L1 proteins, a decrease and a rebound of the amount ofantibodies is observed. Thus, for the first time, not only a diagnosticdetermination of the ad hoc amount of HPV16 L1 capsid specificantibodies is possible but also the detection of a relapse.

When an HPV16 positive primary tumor is treated, within a few weeks(e.g., two weeks), normally in the context of a monitoring, a decreaseof the amount of HPV16 specific L1 antibodies can be observed. Theamount decreases steadily, but levels off at a ground line, so that aplateau is formed (FIG. 1). If HPV16 positive cells of the primary tumorremaining in the body start to grow again (relapse or metastasis), aquick increase of the amount of antibodies is observed (FIG. 2).

This change (rebound) of the amount of antibodies is already observed inthe case of a microscopically small, for a clinician not visiblesecondary tumor, so that at the rebound therapy measures can beinitiated much earlier than commonly done today and the chance ofhealing for the patient increases significantly.

Provided are methods for therapy control of HPV16 positive carcinoma, anantibody for use in the corresponding diagnostic method as well as atest for performing the method. In particular, a serologic method formonitoring the development of the amount of antibodies in samples, whichwere taken from a patient before and after the treatment of a HPV16positive carcinoma over a predetermined period of time is provided. Inaddition, an immunologic test in the form of a kit, with which themethod can be performed, is provided.

Provided is a serologic test, which allows a highly sensitive and typespecific therapy control of HPV16 positive carcinoma and facilitatesdetection of a reoccurrence of the disease, such as a relapse or ametastasis, at an early stage.

Also provided is an in vitro method for therapy control after treatmentof HPV16-positive carcinoma comprising the steps of

(a) contacting a sample from a patient with an HPV16-positive carcinoma,said patient having been administered an anti-cancer therapy, with aplurality of antigens comprising a conformational epitope of HPV16 L1capsid or capsomer, wherein said epitope is not present in monomericand/or denatured HPV16 L1, under conditions at which antibodies presentin the sample can bind to the antigens and

(b) detecting the binding of antibodies in the sample bound to theantigen, wherein the binding of said antibodies bears a negativecorrelation with the success of anti-cancer therapy in the patient.

A sample may be taken from the patient before the treatment or at thetime the anti-cancer therapy is initiated, to obtain a reference valueof the amount of antibodies in the sample, to which later samples can becompared. Antibody levels as high as up to 50,000 ng/ml may be observedat this time in a patient.

In embodiments, the binding of antibodies in the sample bound to theantigen is detected by contacting the sample with labeled antibodiesthat specifically bind to the conformational epitope of the HPV16 L1capsid or capsomer, for example with labeled antibodies obtained fromthe hybridoma cell line with the deposit number DSM ACC3306.

In further embodiments, the binding of said antibodies is compared witha reference level of binding, for example wherein the binding of saidantibodies is compared with the binding of said antibodies in one ormore samples taken from said patient at predetermined time intervals,wherein a decrease in said binding over a predetermined time indicatessuccessful anti-cancer therapy and wherein an increase in said bindingover time indicates a recurrence of the HPV16-positive carcinoma in saidpatient.

The labeled antibodies may be present in mobile form on a test strip andwherein complexes of antigen and labeled antibody are detected bybinding to another antibody, for example wherein the other antibodiesare also ones that are obtained from the hybridoma cell line with thedeposit number DSM ACC3306.

In the described method, a patient identified as having a recurrence ofthe HPV16 positive carcinoma may be administered an anti-cancer therapy.

According to a preferred embodiment, the method for therapy controlafter treatment of HPV16 positive carcinoma comprises or consists of thefollowing steps:

i) Mixing a sample of a patient with a plurality of antigens, whereinthe antigens present HPV16 L1 capsid or capsomer structures, which haveconformational epitopes not present in monomeric and/or denatured HPV16L1, under conditions at which antibodies present in the sample can bindto the HPV16 L1 antigens,

ii) Contacting the mixture of step i) with labeled antibodies, whichspecifically bind to the conformational epitopes of the HPV16 L1 capsidor capsomer structure presenting antigens, particularly with labeledantibodies, which are obtained from the hybridoma cell line with thedeposit number DSM ACC3306,

iii) Quantifying the labeled antibodies and/or the antibodies of thesample, which have bound to the HPV16-L1 capsid or capsomer structurepresenting antigens, respectively,

iv) Repeating steps i) to iii) one or more times with samples taken fromthe same patient in predetermined time intervals so that a trend of theamount of antibodies, that bind to the HPV16 L1 capsid or capsomerstructure presenting antigens, in the patient is tracked based on thesamples over a predetermined period of time, and

v) Determining the amount of antibodies that bind to the HPV16 L1 capsidor capsomer structure presenting antigens in the samples to observe adecrease of the amount after successful therapy, and/or

vi) Determining the amount of antibodies that bind to the HPV16 L1capsid or capsomer structure presenting antigens in the samples toobserve a reoccurrence of a HPV16-positive carcinoma, if the amount ofantibodies that bind to the HPV16 L1 capsid or capsomer structurepresenting antigens increases again in the sample within thepredetermined period of time.

The method allows to quantify the amount of antibodies in the sample ofthe patient over a period of time and thus to track the development ofthe condition after therapy. For example, steps i) to iii) can berepeated every 1 to 4 weeks over a period of one to ten years.

In case of a successful therapy, the amount of antibodies found in thesample of the patient may decrease by over 50% within a couple of weekscompared to the amount of antibodies found before or at the beginning ofthe treatment.

In the method, antigens or virus like particles can be used, whichpresent the HPV16 L1 capsid or capsomer structures, havingconformational epitopes. For example, the conformational epitopesspecifically bind antibodies, which are obtained from the hybridoma cellline with the deposit number DSM ACC3306.

As a sample, a body fluid is suitable, e.g., whole blood or derivativesof whole blood such as, e.g., serum or plasma, as well as capillaryblood or whole blood from the finger pad of the patient. To perform themethod, one drop (approx. 25 μL) is sufficient.

In step i), the sample of the patient can be incubated over a period of1 to 15 minutes, for example, 3 to 10 minutes. Thus, specificinteractions are ensured.

The therapy is in particular a primary therapy by surgery, radiotherapy,chemotherapy or immunotherapy or a combination of these methods.

Typically, in female patients 93% of HPV16-positive carcinoma are foundin the anogenital area and only 7% in the mouth or throat, while in malepatients about 80% are localized in the mouth or throat and only 20% inthe anogenital area.

An HPV16 L1 capsid or capsomer structure is an aggregate or a multimerof the HPV16 L1 protein, which, by interaction of several L1 proteins,forms conformational epitopes on its surface, which are not present inL1 monomers or denatured proteins. In particular, virus like particles(VLPs) are used, which present the HPV16 L1 capsid or capsomerstructures. Some VLPs may carry several of the conformational epitopes,i.e., specific binding sites.

A virus like particle (VLP) is a virus particle, which consists of viralcapsids, but does not contain nucleic acids. The VLPs are thereforesuitable to present the above mentioned conformational epitopes withoutbeing capable of replication.

The labeled antibodies are antibodies directed against the HPV16 L1,which specifically binds to the conformational epitopes of the HPV L1capsid or capsomer structures and thus not to monomeric and/ordentatured HPV16 L1 proteins. A preferred antibody can be obtained fromthe hybridoma cell line, which was deposited under the Budapest Treatyat the Deutsche Sammlung für Mikroorganismen and Zellkulturen (DSMZ),Inhoffenstraße 7B, 38124 Braunschweig, Germany, on 14 Sep. 2016 byAbviris Deutschland GmbH, Ammersbeck, under the deposit number DSMACC3306. For the labeling of the antibody, the skilled person is awareof suitable methods of the state of the art, which allow aquantification of the amount of bound and unbound antibodies. Inembodiments, the antibody is labeled with gold particles.

For the detection, commercially available readers for test strips, e.g.,EseQuant by QIAGEN may be used. The measurement can be performedphoto-optically or by determining the conductivity. Alternatively,colored or radioactively-labeled particles (vesicles) can be used.

The method is absolutely specific for HPV type 16 and there is nocross-reaction with other antibodies. The reason for this is inparticular, that the capsomer structures are present as L1 aggregates,whereby the conformational epitopes are presented on the surface.

When the L1 protein forms aggregates (synonyms are multimers, pentamersor also capsomers), structures (conformational epitopes binding sitesfor antibodies) are formed on the surface (the areas of the capsids thatare oriented towards the outside) of these aggregates by interaction ofseveral L1 proteins, which are specific for particular HPV types (i.e.,type specific).

On the other hand, the highly homologous areas of the L1 protein areoriented such that they are not on the surface but in the inside. Theseareas are necessary for the integration of the viral genetic material.That means, that for the viruses or the non-infectious virus likeparticles (VLPs) used in testing, the above described areas, whichstrongly match within the family of the papilloma viruses, are hiddeninside the particles. These areas are thus no longer accessible in VLPsfor the group specific antibodies in the human samples, whereby it isensured that exclusively type specific antibodies are detectable.

However, monomeric protein cannot be removed by 100% during VLPpurification. Purified VLPs therefore always contain also capsomers.This means, that if these purified L1 proteins are directly immobilizedon a carrier medium (e.g., on classic ELISA plates), both, type specificVLPs but also monomeric L1 protein and capsomers with non-type specificareas are available for testing.

This contamination of the type specific L1 VLPs with monomeric ordentatured protein results in the type specific antibodies in a mixtureof antibodies, such as human samples, being no longer (reliably)identifiable because it cannot be determined whether the test result canbe attributed to the type specific or the group specific binding sites.

The same is true for VLPs, which consist of the L1 and the L2 protein.

Using the approach of competition between the patient antibodies and theHPV16 specific L1 antibodies according to the disclosure, thiscontamination problem can be solved because the test system canexclusively measure HPV16 L1 specific antibodies, which compete with theantibody according to the disclosure.

It is therefore preferred that the antigens are not immobilized but areprovided in a liquid phase, to which the patient sample is added.

This has major advantages or, respectively, the following background: Byimmobilization and the subsequent preservation (by drying loss ofhydrate shell causes conformational changes of the protein) required forselling the ELISA plates, the antigens (VLPs) are changed, i.e., theylose their type specific epitopes and thus become useless for testing.When adding the patient sample to the VLP solution, the antibodies ofthe patient sample and the antigen come into close proximity. Thus, thekinetics of the binding reaction become significantly faster, which isextremely useful for a quick test. Furthermore, the analytic sensitivityis increased because all antibodies in the patient sample are “quickly”available for the testing.

When immobilized on a carrier material, e.g., a microtiter plate, on theother hand, only the antibodies in close proximity to the surface areavailable as reactants. The antibodies within the lumen of the reactionvessel will practically “never” reach the surface of the reaction vesselbecause they have to cross the distance of 1-2 mm to the surfaceexclusively by Brownian motion, which takes “a lot” of time.

Described is also a method as described above, wherein the patientsample is simultaneously mixed with the antigens and contacted with thelabeled antibodies.

Advantageously, it is also possible to simultaneously mix the patientsample with the antigens and contact it with the antibodies. This way, adirect competition between the antibodies from the patient sample andthe labeled antibodies for the binding sites is achieved, which due tothe fast binding kinetics as described above leads to more accurate testresults. Accordingly, the test method can be performed in one step.

Further described is a method as described above, wherein in step ii)the mixture runs across a test strip, on which the labeled antibodiesare present in mobile form and wherein in step iii) complexes of antigenand labeled antibody are detected by binding to another antibody,wherein preferably the other antibodies are also ones that are obtainedfrom the hybridoma cell line with the deposit number DSM ACC3306.

This embodiment allows to provide a quick test, in which in a reactionzone, a quickly readable test result line becomes visible.

The mixture of step i) is applied at an application area on the teststrip and then runs, for example by using capillary forces, across amembrane, during which it comes into contact with the labeled antibodieson a stretch up to the reaction zone. The labeled antibodies areconcentrated such that not all antigens or binding sited are filled up.In the reaction zone, further antibodies are immobilized, which are alsospecific for the antigens or binding sites described herein and bind thelabeled antibody-antigen complexes, insofar as free binding sites arestill available. Thus, the labeled antibody-antigen complexes areretained in the reaction zone, whereby a test line becomes visible. Incase the antigens or binding sites, respectively, are already occupiedby antibodies from the patient sample, due to the competitive approach,less antibody-antigen complexes are formed with the labeled antibodiesand the test line is less intense or not visible at all, that is, incase all binding sites were already occupied by the antibodies from thepatient sample at the time of application. The correct adjustment of theamount of antigens or binding sites, respectively, in step i) andlabeled antibodies in step ii) is important in this context, so that achange in the amount of antibodies in the patient sample with respect toa previous measurement can be detected.

Described is a method of treating a patient that has previously beenadministered at least one anti-cancer therapy targeting anHPV16-positive carcinoma comprising (A) requesting a test providingresults of an analysis to determine whether the patient has an increasein antibodies that bind to a conformational epitope of HPV16 L1 capsidor capsomer over a predetermined time period and (B) administering anadditional anti-cancer therapy targeting HPV16-positive carcinoma if anincrease in antibodies is detected in the patient.

Further described is an antibody, which specifically binds toconformational epitopes of HPV16-L1 capsid or capsomer structurepresenting antigens, for example an antibody, which is obtained from thehybridoma cell line with the deposit number DSM ACC3306.

Also described is an antibody as described above for use in a diagnosticmethod, in particular in a method for determining the reoccurrence of aHPV16-positive carcinoma after treatment.

Moreover, described is an antigen, which presents HPV16 L1 capsid orcapsomer structures or a virus like particle, which presents the HPV16L1 capsid or capsomer structures.

Described is an antigen or a virus like particle, which presents HPV16L1 capsid or capsomer structures having conformational epitopes thatspecifically bind an antibody obtained from the hybridoma cell line withthe deposit number DSM ACC3306.

An antigen or a virus like particle as described above for use in adiagnostic method, in particular in a method for determining thereoccurrence of a HPV16-positive carcinoma after treatment is alsodescribed. Further described is a kit for determining an amount ofantibodies in a sample of a patient comprising or consisting of:

I) a composition comprising antigens presenting conformational epitopesof HPV16 L1 capsid or capsomer structures, and

II) a composition comprising labeled antibodies, which specifically bindto conformational epitopes of HPV16 L1 capsid or capsomer structurepresenting antigens, wherein preferably the labelled antibodies areobtained from the hybridoma cell line with the deposit number DSMACC3306.

The composition i) is preferably a composition, which comprises viruslike particles, which present conformational epitopes of HPV16 L1 capsidor capsomer structures.

A kit can also comprise a test strip, which optionally is provided in atesting cassette, which has openings for applying the mixture of step i)as well as for observing a test line and preferably a control line. Thetest strip comprises on one end an application zone for the mixture ofstep i), a pad, on which the labeled antibodies are provided and whichconnects to the application zone, as well as a reaction zone, whichconnects on the other side of the pad when viewed from the applicationzone and preferably a control zone, further beyond the reaction zone. Inthe reaction zone, the further antibody is provided, which specificallybinds the epitope described herein and by binding of the labeledantibody-antigen complexes makes the test line visible. Anotherindependent antibody reaction in the control zone shows that the testproceeded correctly. The appearance of a line in the control zoneconfirms that the sample volume was sufficient and the test ran asintended.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the development of the amount of antibodies in the serum ofa patient with a positive therapy course over 27 weeks after primarytherapy of a HPV16 positive carcinoma. On the X axis, the weeks startingfrom 0 at the point in time of the primary therapy are shown, on the Yaxis, the concentration of the antibody is shown in ng/ml.

FIG. 2 shows the development of the amount of antibodies in the serum ofa patient, in which after about 35 weeks after the primary therapy of aHPV16 positive carcinoma, a relapse occurred. Up to week 27, acontinuous decrease of the amount of antibodies was observed. Beginningin week 31, the amount of antibodies increased again. The increasecontinued until week 35, at which time the clinical correlation wasfound. On the X axis, the weeks starting from 0 at the point in time ofthe primary therapy are shown, on the Y axis, the concentration of theantibody is shown in ng/ml.

DETAILED DESCRIPTION Example 1 Screening for the Antibodies and Antigens

Preparation of papillom virus like particles (VLPs): The L1 gene ofHPV16 (GenBank: K02718.1) was amplified by PCR and cloned into thetransfer vector pVL1392. The recombinant vectors were introduced in Sf9cells together with BaculoGold DNA (Pharmingen) using calcium phosphateprecipitation. Recombinant viruses were amplified and purified by plaqueassay according to manufacturer's instructions.

Virus like particles (VLPs) were purified according to Volpers et al.(Volpers, C., P. Schirmacher, R. E. Streeck, and M. Sapp. 1994. Assemblyof the major and the minor Kapsid protein of human papilloma virus type33 into virus-like particles and tubular structures in insect cells.Virology 200:504-512).

Production, screening and cloning of the monoclonal antibodies. BALB/cmice were subcutaneously immunized with 20 μg of intact HPV16 VLPsdissolved in phosphate buffered salt solution (PBS), after these hadbeen mixed with complete Freund's adjuvant. The immunization wasrepeated after one month and after three months.

Three days after the third immunization the spleen was taken out and asingle cell suspension was produced. The spleen cells were fused withthe mouse myeloma cell line X63Ag8.653 using polyethylene glycol 2500(Boehringer Mannheim) and cultured in Iscoves modified Eagle Medium(IMDM) in the presence of 10% fetal calf serum in 96-well plates. Fusedcells were selected with azaserine and hypoxanthine. After 6 to 8 days,the supernatant of the cells was tested for secretion of HPV16 L1specific antibodies using ELISA. Denatured L1 protein, as well as VLPsof HPV-6, HPV-11, HPV-18, HPV-31, HPV-33 and HPV-39 served as controlsto exclude unspecific reactivities.

Example 2 Observation of the Decrease of the Amount of Antibodies in aPatient after Successful Therapy

Male Patient, age 53, with oncologic combination therapy(surgery/radio-chemotherapy) for a HPV16 positive tonsillar carcinoma.On the day before the therapy began, 5 ml blood were taken from thepatient to obtain patient serum. Testing of the serum at the beginningof the therapy gave an antibody concentration of 13200 ng/ml.

Six weeks after the primary therapy, 5 ml blood were taken from thepatient again to obtain serum. An antibody concentration of 5600 ng/mlwas measured. This corresponds to a decrease of the antibodyconcentration of over 50% within 6 weeks.

With the decrease of the amount of antibodies, a successful therapy canbe controlled because the tumor antigen HPV16 L1 forming tumor cellswere successfully removed and the tumor antigen (HPV16 L1 protein) doesnot induce the immune system anymore to form HPV16 L1 specificantibodies.

The invention claimed is:
 1. A method for continued treatment ofHPV16-positive carcinoma in a patient, the method comprising: (a)contacting a sample from the patient with an HPV16-positive carcinoma,said patient having been administered an anti-cancer therapy, with aplurality of antigens comprising a conformational epitope of HPV16 L1capsid or capsomer, wherein said epitope is not present in monomericand/or denatured HPV16 L1, under conditions at which antibodies presentin the sample can bind to the antigens; (b) detecting antibody levels inthe sample that bind to the antigens; (c) comparing the antibody levelsto a reference level of antibodies, wherein the reference level ofantibodies is from a sample taken from the patient prior to theanti-cancer therapy, wherein a reduction in the antibody levels isindicative the anti-cancer therapy in the patient is working, andwherein an increase in the antibody levels is indicative the anti-cancertherapy in the patient is not working; and (d) administering anadditional anti-cancer therapy targeting HPV16-positive carcinoma in thepatient, wherein the patient has an increase in the antibody levelscompared to the reference level of antibodies.
 2. The method accordingto claim 1, wherein detecting antibody levels in the sample includescontacting the sample with labeled antibodies that specifically bind tothe conformational epitope of the HPV16 L1 capsid or capsomer.
 3. Themethod according to claim 2, wherein the labeled antibodies are presentin mobile form on a test strip, and wherein complexes of the antigen andthe labeled antibody are detected by binding to a subsequent antibody.4. The method according to claim 1, wherein the patient is identified ashaving a recurrence of the HPV16 positive carcinoma.
 5. A method forcontinued treatment of HPV16-positive carcinoma in a patient, the methodcomprising: i) mixing a sample of the patient with a plurality ofantigens, said patient having been administered an anti-cancer therapy,wherein the antigens present HPV16 L1 capsid or capsomer structures,which have HPV16 L1 conformational epitopes not present in monomericand/or denatured HPV16 L1, under conditions at which antibodies presentin the sample can bind to the HPV16 L1 antigens, ii) contacting themixture of step i) with labeled antibodies, which specifically bind tothe conformational epitopes of the HPV16 L1 capsid or capsomer structurepresenting antigens, iii) quantifying a level of the labeled antibodiesand/or the antibodies of the sample, which have bound to the HPV16 L1capsid or capsomer structure presenting antigens, respectively, iv)repeating steps i) to iii) one or more times with samples taken from thesame patient in predetermined time intervals so that a trend of thelevel of antibodies that bind to the HPV16 L1 capsid or capsomerstructure presenting antigens in the patient is tracked based on thesamples over a predetermined period of time, v) determining the level ofantibodies that bind to the HPV16 L1 capsid or capsomer structurepresenting antigens in the samples, vi) comparing the level ofantibodies determined in step v) with a reference level of antibodies,wherein the reference level of antibodies is from a sample taken fromthe patient prior to the anti-cancer therapy, wherein a decrease of thelevel of antibodies that bind over the predetermined period of timeindicates the therapy is working, and wherein an increase in the levelof antibodies that bind over the predetermined period of time indicatesa reoccurrence of the HPV16-positive carcinoma, and vii) administeringan additional anti-cancer therapy targeting HPV16-positive carcinoma inthe patient, wherein the patient has an increase in the antibody levelscompared to the reference level of antibodies.
 6. The method accordingto claim 1, wherein the antigens are not immobilized but are provided ina liquid phase, to which the patient sample is added.
 7. The methodaccording to claim 2, wherein the patient sample is simultaneouslycontacted or mixed with the antigens and contacted with the labeledantibodies.
 8. The method according to claim 5, wherein in step ii) themixture runs across a test strip, on which the labeled antibodies arepresent in mobile form, and wherein in step iii) complexes of antigenand labeled antibody are detected by binding to another antibody.
 9. Themethod according to claim 2, wherein the labeled antibodies are obtainedfrom the hybridoma cell line with the deposit number DSM ACC3306. 10.The method according to claim 1, wherein the reference level ofantibodies are a plurality of reference levels of antibodies taken fromthe patient at predetermined time intervals.
 11. The method according toclaim 3, wherein the subsequent antibody is one that is obtained fromthe hybridoma cell line with the deposit number DSM ACC3306.
 12. Themethod according to claim 8, wherein the another antibody is obtainedfrom the hybridoma cell line with the deposit number DSM ACC3306. 13.The method according to claim 5, wherein the labeled antibodies areobtained from the hybridoma cell line with the deposit number DSMACC3306.